Genetics Lecture 3 Final (2024) PDF

11/13/2023 General Microbiology and Immunology Bacterial Genetics Lecture 3 Lecture outline Genetic engineering, molecular cloning, recombinant DNA (rDNA) technology and biotechnology. Some basic tools of genetic...

11/13/2023 General Microbiology and Immunology Bacterial Genetics Lecture 3 Lecture outline Genetic engineering, molecular cloning, recombinant DNA (rDNA) technology and biotechnology. Some basic tools of genetic engineering: Restriction enzymes Cloning vectors Polymerase chain reaction. Agarose gel electrophoresis. 1  11/13/2023 Manipulation of DNA Genetic engineering refers to the use of in vitro techniques to alter genes in the lab. These techniques (also called molecular cloning/ recombinant DNA technology) include those for the isolation, purification, manipulation, amplification/replication and expression of genes efficiently in host organisms (mainly microorganisms). These molecular techniques are the foundation of biotechnology. P.S. in vivo cloning has many limitations. Biotechnology is the use of microorganisms (genetically modified), cells, or cell components to make a product (such as food, vaccine, antibiotic, vitamins…) for industrial, medical or agricultural applications. In most cases, microorganisms and plants are being used as “factories” to produce chemicals that the organisms don’t naturally make. HOW? By inserting, deleting, or modifying genes with recombinant DNA (rDNA) technology, which is sometimes called genetic engineering. The recipient can then be made to express the gene which may code for a commercially useful product. e.g. Insulin (gene from human…into…> bacteria). e.g. Hepatitis B vaccine (gene encoding viral coat protein…into…> yeast) (advantage of genetic recombinant vaccine over conventional vaccines). Remember: recombination of DNA naturally occurs in microbes. In 1970s-1980s, scientists developed artificial techniques for making rDNA. 2  11/13/2023 An overview of recombinant DNA procedures Some tools of rDNA technology Restriction endonucleases Vectors Polymerase chain reaction 3  11/13/2023 Restriction endonucleases Recombinant DNA technology has its technical roots in the discovery of restriction endonucleases (restriction enzymes), a special class of DNA cutting enzymes that exist in many bacteria, however, they are very rare in eukaryotes. First isolated in 1970, restriction enzymes in nature had actually been observed earlier, when certain bacteriophages were found to have a restricted host range. If these phages were used to infect bacteria other than their usual hosts, restriction enzymes in the new host destroyed almost all the phage DNA. Restriction enzymes protect a bacterial cell by hydrolyzing phage DNA. Restriction enzymes are used for in vitro DNA manipulation and are a major tool of genetic engineering. Mechanism of Restriction Enzymes Restriction enzymes recognize specific base sequences (recognition sequences) within DNA and cut the phosphodiester backbone, resulting in double-stranded breaks. Restriction endonucleases are divided into three major classes. Type I and III restriction enzymes bind to the DNA at their recognition sequences but cut the DNA at some distance away. In contrast, the type II restriction enzymes cleave the DNA within their recognition sequences, making this class of enzymes much more useful for the specific manipulation of DNA. Most of the DNA sequences recognized by type II restriction enzymes are short inverted repeats of 4, 6 or 8 base pairs (bp). (palindromes) 4  11/13/2023 Mechanism of Restriction Enzymes Hundreds of restriction enzymes are known, each producing DNA fragments with characteristic ends. They are named for their bacterial source. Some of these enzymes (e.g., HaeIII) cut both strands of DNA in the same place, producing blunt ends, and others make staggered cuts in the two strands—cuts that are not directly opposite each other. These staggered ends, or sticky ends, are most useful in rDNA because they can be used to join two different pieces of DNA that were cut by the same restriction enzyme. Staggered cuts leave stretches of single-stranded DNA at the ends of the DNA fragments. 5  11/13/2023 If two fragments of DNA from different sources have been produced by the action of the same restriction enzyme, the two pieces will have identical sets of sticky ends and can be spliced (recombined) in vitro. The sticky ends join spontaneously by hydrogen bonding (complementary base pairing). The enzyme DNA ligase is used to covalently link the backbones of the DNA pieces, producing an rDNA molecule. Protection from Restriction?? The natural role of restriction enzymes is to protect the cell from invasion by foreign DNA, especially viral DNA. If foreign DNA enters the cell, the restriction enzymes will destroy it. However, a cell must protect its own DNA from destruction by its own restriction enzymes. Such protection is conferred by modification enzymes. Each restriction enzyme is partnered with a corresponding modification enzyme that shares the same recognition sequence. The modification enzymes chemically modify specific nucleotides in the restriction recognition sequences of the cell’s own DNA. These modified sequences can no longer be cut by the corresponding restriction enzymes. Typically, modification consists of methylating specific bases within the recognition sequence, which prevents the restriction endonuclease from binding. If even a single strand is modified, the recognition sequence is no longer a substrate for the corresponding restriction enzyme. 6  11/13/2023 Cloning vectors Cloning vectors Cloning vectors are small, independently replicating genetic elements used to carry and replicate the desired cloned DNA segments. The resulting recombinant vector is transformed into a host cell for replication; i.e. cloning. Most vectors are plasmids or viral DNA. Plasmid’s circular form protects it from being destroyed by its recipient. Plasmid DNA can be cut with the same restriction enzymes as the DNA that will be cloned, so that all pieces of the DNA will have the same sticky ends. When the pieces are mixed, the DNA to be cloned will be inserted into the plasmid. Note that another possible option is the self-ligation of the plasmid without the target DNA insert. 7  11/13/2023 Cloning vectors A different kind of vector is viral DNA. This type of vector can usually accept much larger pieces of foreign DNA than plasmids can. After the DNA has been inserted into the viral vector, it can be cloned in the virus’s host cells. The DNA of the virus inserts itself quickly into the chromosome of the host, so as to evade any destruction by the host cell. Retroviruses, adenoviruses, and herpesviruses are being used to insert corrective genes into human cells that have defective genes. (later in gene therapy) When it is necessary to retrieve cells that contain the vector, a marker gene in the vector often helps make selection easy. Common selectable marker genes are for antibiotic resistance or for an enzyme that carries out an easily identified reaction. Steps in gene cloning 1. Isolation and fragmentation of the source DNA: total genomic DNA from an organism of interest, gene/genes amplified by PCR or synthetic DNA made in vitro. If genomic DNA is the source, it is first cut with restriction enzymes to give a mixture of fragments of manageable size. 2. Inserting the DNA fragment into a cloning vector. 3. Introduction of the cloned DNA into a host organism where it can replicate. In practice this often yields a mixture of recombinant constructs. Some cells contain the desired cloned gene, whereas other cells may contain other cloned genes from the source DNA. 8  11/13/2023 Genomic library Genomic library: Different clones, each containing different cloned DNA segments from the source organism. Selecting the right clone?? - Antibiotic resistance. - DNA sequencing. - Hybridization. Polymerase chain reaction (PCR) PCR is essentially DNA replication in vitro. In few hours, PCR can copy segments of DNA by up to a billionfold in the test tube, a process called amplification. This yields large amounts of specific genes or other DNA segments that may be used for a range of applications in molecular biology. 9  11/13/2023 PCR principle PCR uses the enzyme DNA polymerase, which naturally copies DNA molecules. PCR does not actually copy whole DNA molecules but amplifies stretches of up to a few thousand base pairs (the target) from within a larger DNA molecule. Artificially synthesized oligonucleotide primers are used to initiate DNA synthesis, but they are made of DNA (rather than RNA primers used by cells). Primers are chosen to flank (be placed at both sides of) the targeted DNA region. PCR cycles The reaction mixture consists of: 1. DNA template. 2. The two oligonucleotide primers (FOR and REV, 18-30 nucleotides each). 3. DNA polymerase. 4. Mixture of all four deoxynucleotides (A,C,G&T). The mixture is heated once at 95°C for 5 minutes to initialize the reaction. 10  11/13/2023 PCR cycles Each PCR cycle consists of: 1. Denaturation step: The mixture is heated to 95°C for 30 seconds. 2. Annealing step: The temperature is then lowered to allow the primer to anneal/hybridize with the complementary regions on target DNA. This step occurs at 50-65°C for 30 seconds. P.S. Primers are added in excess to ensure that most template strands anneal to a primer and not to each other. 3. Primer extension (elongation) step: DNA polymerase (Taq polymerase) extends the primers using the target strands as template. This step occurs at 72°C for 1 minute/1000 bases polymerized. Then this cycle is repeated. In practice, 20-30 cycles are usually run yielding 106-109 fold increase in the copies of the target sequence. PCR tubes Thermocycler: A machine that can be programmed to run through heating and cooling cycles automatically. 11  11/13/2023 PCR (annealing temperature) The annealing temperature is a key variable in determining the specificity of a PCR protocol This temperature depends on the length and sequences of the primers Annealing temperature=Tm- (2 to 5° C) Tm: melting temperature (specific for the primer). Tm = 4 (G+C) + 2 (A+T) DNA polymerase in PCR Because high temperatures are used to denature the double-stranded copies of DNA in vitro, a thermostable DNA polymerase isolated from the thermophilic hot spring bacterium Thermus aquaticus is used. DNA polymerase from T. aquaticus, called Taq polymerase, is stable to 95°C and thus is unaffected by the denaturation step employed in the PCR. DNA polymerase from Pyrococcus furiosus, a hyperthermophile with a growth temperature optimum of 100°C is called Pfu polymerase and is even more thermostable than Taq polymerase. Moreover, unlike Taq polymerase, Pfu polymerase has proofreading activity, making it especially useful when high accuracy is crucial. 12  11/13/2023 PCR PCR is a powerful tool and has revolutionized different fields of biology. It is easy to perform, extremely sensitive, specific, and highly efficient. During each round of amplification the amount of product doubles, leading to an exponential increase in the DNA. Number of copies of the target sequence= 2n n= number of PCR cycles Variations in the standard PCR procedure 1- Reverse transcription PCR (RT-PCR) can be used to make DNA from viral RNA or an mRNA template. This procedure can be used to detect if a gene is expressed or to produce an intron-free eukaryotic gene for expression in bacteria (as in case of recombinant insulin). RT-PCR uses the reverse transcriptase enzyme to convert RNA into complementary DNA (cDNA) which is then amplified. 13  11/13/2023 Variations in the standard PCR procedure (cont.) 2- Real-time PCR or quantitative PCR (qPCR) is the technique of collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step. qPCR uses fluorescent dyes or fluorescent probes to monitor the amplification process and quantify the amount of initial target DNA in a sample. 14  11/13/2023 Applications of PCR 1. Synthesis of DNA for cloning and sequencing. 2. Identification of isolated genes. 3. Diagnosis of infectious and non-infectious diseases. 4. DNA fingerprint in forensic medicine. Agarose gel electrophoresis 15  11/13/2023 Electrophoresis is a procedure that separates charged molecules by migration in an electrical field. The rate of migration is determined by the charge on the molecule and by its size. In vitro manipulation of nucleic acid often requires separation of molecules based on size. Agarose gel electrophoresis is an essential step during molecular cloning where it is used to separate, identify, and purify DNA fragments. For example, many restriction enzymes cut DNA molecules into segments that range in length from a few hundred to a few thousand base pairs. After the DNA is cleaved, the fragments generated can be separated from each other by gel electrophoresis and analyzed. Gel electrophoresis is also used to verify that amplification of a nucleic acid was successful. Load the gel with samples and ladder Poured gel containing ethidium bromide UV visualization 16  11/13/2023 DNA Marker (Ladder) Agarose is a linear polymer composed of alternating residues of D- and L- galactose joined by glycosidic linkages. Gelation of agarose results in a three-dimensional mesh of channels whose diameters range from 50 nm to >200 nm. When DNA is loaded in the agarose gel and upon application of electric current, the negatively charged DNA will migrate towards the positive electrode. Small size DNA fragments will migrate faster than large size DNA fragments so separation occurs. Size of DNA can be approximately determined by running a standard DNA ladder (DNA marker) in parallel. 17  11/13/2023 The location of DNA within the gel can be determined directly by staining with low concentrations of fluorescent intercalating dyes, such as ethidium bromide. DNA can be detected by direct examination of the gel in UltraViolet (UV) radiation. If necessary, these bands of DNA can be extracted from the gel and used for different applications. 18  11/13/2023 Factors affecting the rate of migration of DNA in agarose: 1- Double-stranded DNA migrate through gel matrices at rates that are inversely proportional to the log10 of the number of base pairs. Larger molecules migrate more slowly because of greater frictional drag and because they worm their way through the pores of the gel less efficiently than smaller molecules. Factors affecting the rate of migration of DNA in agarose: 2- The concentration of agarose: Migration of DNA fragments becomes at slower rates in gels containing higher concentrations of agarose. The concentrations of agarose usually used range from 0.5-2%. 3- The applied voltage: The rate of migration of linear DNA fragments increases by increasing the applied voltage. 19  11/13/2023 Thank you 20